The salting-out effect describes a drop in solute solubility, especially for large biomolecules like proteins and oligonucleotides, once the ionic strength of an aqueous solution climbs very high (1). Chemists have relied on this behavior for well over a century before anyone fully understood what salting out is at a molecular level.

Analysts turn to this shift in solubility because it gives them a controlled, reversible way to isolate proteins, oligonucleotides, and other biomolecules without harsh chemical treatment. Labs also lean on this behavior to clean crude extracts and drive selective binding inside a chromatography column. Salting out remains one of the most dependable tools a method developer has for separating complex biological mixtures.

Salting out is the phenomenon where adding a high concentration of a soluble salt to an aqueous solution reduces the solubility of dissolved solutes, often forcing them to precipitate or partition into a separate phase (1). Adding a highly soluble salt, most often ammonium sulfate, raises the ionic strength of the buffer until water molecules preferentially cluster around the salt ions rather than the solute. The solute then has two options: precipitate out of the aqueous phase entirely, or partition onto a nearby hydrophobic surface, which is exactly what happens inside a chromatography column.

This process differs sharply from salting in, where a low to moderate salt concentration actually increases solute solubility by shielding charged patches on the protein surface and preventing self-aggregation (2). Kosmotropic salts drive salting out, while chaotropic salts drive salting in, and the anion type in a given salt matters far more than the cation. Method developers who understand the difference between salting out and salting in can pick the right salt and concentration for their separation goal.

Understanding how salting out works starts with water itself. In pure water, proteins and other partially hydrophobic solutes sit inside an ordered shell of water molecules, sometimes called a hydration shell or water cage, that keeps hydrophobic patches shielded and the solute dissolved. Add a high concentration of a salting-out salt, and the salt ions compete aggressively with the protein for that same water of solvation (3). The ions pull water molecules away from the protein surface, and the hydration shell breaks down. Once that shell disappears, hydrophobic patches on the protein surface become exposed to the bulk solvent, and the system responds by minimizing free energy, driving those exposed hydrophobic surfaces to associate with each other or with a hydrophobic ligand nearby.

This salting out mechanism is entropy-driven and follows the second law of thermodynamics, since the system moves toward the state with the least ordered water. That single principle explains why salting out triggers protein precipitation in bulk solution and protein binding onto a hydrophobic interaction chromatography column at the same time.

Understanding how salting out works starts with water itself. In pure water, proteins and other partially hydrophobic solutes sit inside an ordered shell of water molecules, sometimes called a hydration shell or water cage, that keeps hydrophobic patches shielded and the solute dissolved. Add a high concentration of a salting-out salt, and the salt ions compete aggressively with the protein for that same water of solvation (3). The ions pull water molecules away from the protein surface, and the hydration shell breaks down. Once that shell disappears, hydrophobic patches on the protein surface become exposed to the bulk solvent, and the system responds by minimizing free energy, driving those exposed hydrophobic surfaces to associate with each other or with a hydrophobic ligand nearby.

This salting out mechanism is entropy-driven and follows the second law of thermodynamics, since the system moves toward the state with the least ordered water. That single principle explains why salting out triggers protein precipitation in bulk solution and protein binding onto a hydrophobic interaction chromatography column at the same time. The Hofmeister series ranks ions by how strongly they stabilize or destabilize proteins in solution, and chemists have used this ranking to pick effective salting out salts for well over a century (4). Ions near the kosmotropic end of the series, small and highly charged species like sulfate, phosphate, and ammonium, hold onto water tightly and pull it away from protein surfaces, which drives salting out and precipitation. Ions near the chaotropic end, larger and less charged species like thiocyanate and perchlorate, interact more weakly with water and actually increase protein solubility, producing salting in instead.

Ammonium sulfate salting out remains the default choice for most protein workflows because ammonium sulfate pairs a strongly kosmotropic anion with excellent aqueous solubility, letting analysts reach very high ionic strength without hitting the solubility limit of the salt itself. Sodium sulfate and potassium phosphate offer similar kosmotropic strength but dissolve less readily, which is why ammonium sulfate still dominates both precipitation protocols and hydrophobic interaction chromatography mobile phases.

Hydrophobic interaction chromatography (HIC) is the clearest real-world example of this mechanism at work, using one of the gentler protein-purification methods available today. A sample gets loaded onto a hydrophobic stationary phase, commonly a C4, butyl, phenyl, or ether ligand, while the mobile phase carries a high salt concentration, often 1.5–2 M ammonium sulfate (5). That high ionic strength strips the ordered water layers away from both the protein and the ligand surface at once, exposing hydrophobic patches on each side and letting the protein bind reversibly to the column.

Because the interaction depends entirely on salt concentration, elution happens through a descending salt gradient rather than a change in pH or an organic solvent wash. As the salt concentration drops step by step, water molecules reform their ordered shells around the protein and the ligand, the hydrophobic interaction weakens, and each protein leaves the column in order of increasing surface hydrophobicity. This behavior sits at the opposite end of the spectrum from ion exchange chromatography (IEX), where a rising salt concentration causes elution instead of binding. That contrast is exactly why HIC often runs directly after an ion exchange step, or right after an ammonium sulfate precipitation cut, with no buffer exchange needed between the two.

Analysts running HPLC-based HIC methods should recognize that salting out HIC and ion exchange chromatography use the same variable, salt concentration, to produce opposite outcomes, which makes the two techniques natural partners in a multi-step purification train.

The salting-out effect shows up across protein purification, small-molecule extraction, and sample cleanup in labs that never touch a HIC column directly.

Hydrophobic Interaction Chromatography

HIC remains the most direct chromatographic use of the salting-out effect, converting a solubility problem into a reversible binding event on a hydrophobic column bed (6). Analysts favor HIC for monoclonal antibodies and other biologics because the high-salt environment preserves native protein folding far better than organic solvents or extreme pH shifts.

Protein Precipitation and Ammonium Sulfate Cuts

Long before HIC columns existed, biochemists used ammonium sulfate cuts to fractionate crude protein mixtures by adding salt in stages and collecting whatever precipitates at each cut (1). Different proteins reach their solubility limit at different ammonium sulfate concentrations, so a 30% cut pulls down one set of proteins while a 60 percent cut pulls down another, giving a crude but useful first purification step before finer chromatography methods take over.

Salting-Out Assisted Liquid–Liquid Extraction and QuEChERS

Salting-out assisted liquid–liquid extraction (SALLE) applies the same solubility shift to organic solvent partitioning, adding salt to a water-miscible solvent like acetonitrile until it splits into two distinct phases (7). Analysts use SALLE as a faster, lower-solvent alternative to traditional liquid–liquid extraction for pulling drugs and metabolites out of plasma or other biological fluids.

QuEChERS, a related method built for food and environmental testing, uses magnesium sulfate to partition pesticide residues away from water during sample cleanup, following the same salting-out logic before the extract moves on to LC or GC analysis (8).

Removing Salts After Salting Out: Desalting Techniques

Every salting-out step leaves the sample loaded with salt, so removing that salt becomes the next practical task for the analyst.

• Solid-Phase Extraction (SPE)-Based Desalting: Passes the sample through a solid sorbent bed that retains the analyte while salts wash through, a standard step before mass spectrometry analysis (9).
• LC Column-Based Desalting: Relies on a size-exclusion spin column to separate small salt ions from larger biomolecules based on molecular size alone (10).
• Dialysis and Ultrafiltration: A semi-permeable membrane is used with a defined molecular weight cutoff, letting salt ions diffuse or get pushed out while the target biomolecule stays behind (10).

Classical Salting-Out Chromatography of Organic Non-electrolytes

Salting out is not limited to proteins. Chemists have long used Hofmeister-informed salt selection to extract organic non-electrolytes, uncharged small molecules that partition based on hydrophobicity rather than charge, from aqueous process streams (4).

Choosing a strongly kosmotropic salt like sodium sulfate over a weaker one can improve extraction yield for these compounds without changing anything else in the process.

Whether the target is a protein or a small organic molecule, every salting-out decision ultimately traces back to where a given salt sits on the Hofmeister series.

Salting-out salts are small, highly charged kosmotropes that pull water away from the solute, decrease solubility, strengthen hydrophobic interactions, and generally preserve native protein structure, which is why HIC binding happens under high salt.

Salting-in salts are larger, weakly charged chaotropes that increase solubility, weaken hydrophobic interactions, and can denature protein structure at high enough concentration, which is why the same conditions elute rather than bind a protein in HIC.

The table below summarizes the practical differences an analyst needs when choosing a salt for a given step.

A few habits keep salting-out methods reliable and protect expensive HPLC hardware over time.

• Filter every high-salt buffer through a 0.22 µm membrane before use to keep particulates and undissolved salt crystals out of the pump and column (3).
• Watch backpressure closely, since high-salt mobile phases run noticeably more viscous than standard aqueous buffers.
• Flush the column with low-salt water, then a storage solvent, at the end of every run to prevent salt crystallization inside the column bed.
• Match column chemistry to analyte hydrophobicity: C4 for the most hydrophobic proteins, butyl or phenyl for moderate hydrophobicity, and ether ligands for the most delicate, least hydrophobic targets.
• Start method development with ammonium sulfate before testing alternative salts, since it offers the best combination of solubility and kosmotropic strength for most proteins.

What salt concentration is typically used in HIC?

Most HIC methods start with 1.5–2 M ammonium sulfate, though some protocols push closer to 2.4 M depending on the protein and column chemistry. The exact starting concentration depends on how hydrophobic the target protein is and how strongly the stationary phase ligand binds. Method developers usually screen a few concentrations before settling on a final gradient.

How Can Salt Be Removed After Salting Out?

Analysts typically choose SPE, size-exclusion spin columns, or membrane-based dialysis and ultrafiltration depending on sample volume and throughput needs. Each method separates salt from the target biomolecule using a different physical principle: sorbent retention, molecular size, or membrane pore size. The right choice depends on how much sample volume the lab can spare and how fast the next analytical step needs the desalted sample.

Can salting out be used for small molecules or only proteins?

Salting out works on small organic non-electrolytes as well as large biomolecules, since the effect depends on hydrophobicity and water structure rather than molecular size alone. SALLE and QuEChERS both apply salting-out principles to small-molecule extraction in bioanalysis and food testing. Proteins simply show the effect more dramatically because of their larger, more variable hydrophobic surfaces.

Is HIC the same as salting out?

HIC is a chromatographic technique that applies the salting-out effect, but the two terms are not interchangeable. Salting out describes the underlying physical phenomenon, disrupted hydration shells and exposed hydrophobic surfaces, while HIC is one specific method that uses that phenomenon to separate proteins on a column. Ammonium sulfate precipitation and SALLE rely on the same phenomenon without ever touching a chromatography column.

1. National Center for Biotechnology Information. (n.d.). Protein precipitation using ammonium sulfate. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC4817497/
2. National Center for Biotechnology Information. (n.d.). Anion binding to hydrophobic concavity is central to the salting-in effects of Hofmeister chaotropes. PMC. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098002/
3. Ewonde, R. E., et al. (2022). A protocol for setting-up robust hydrophobic interaction chromatography. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC10989582/
4. American Chemical Society. (2018). General principles and strategies for salting-out informed by the Hofmeister series. Organic Process Research & Development. https://pubs.acs.org/doi/10.1021/acs.oprd.7b00197
5. National Center for Biotechnology Information. (2011). Automated hydrophobic interaction chromatography column screening. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC3230179/
6. MadSci Network. (2008). What is the mechanism of 'salting-out' or 'salting-in'. https://www.madsci.org/posts/archives/2008-04/1208150541.Bc.r.html
7. National Center for Biotechnology Information. (2023). Salting-out assisted liquid–liquid extraction combined with LC-MS/MS for organic UV filters in environmental water samples. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC10567945/
8. National Library of Medicine. (2011). QuEChERS sample preparation approach for mass spectrometric analysis of pesticide residues in foods. PubMed. https://pubmed.ncbi.nlm.nih.gov/21643905/
9. National Institute of Immunology. (n.d.). Sample desalting procedure [SOP]. https://www.nii.res.in/pdf/SOPs%20for%20Desalting%20procedure.pdf